The present invention relates to methods of treating or preventing EBV infections. The present invention also relates to cytotoxic T-cell (CTL) epitopes within Epstein-Barr virus (EBV) structural and latent antigens, and to subunit vaccines and nucleic acid vaccines which include these epitopes.
It is now well established that long-term protection from persistent viral infection requires the development of virus-specific memory T cells which recognize viral antigens in association with either class I or class II MHC molecules. Since immunization with whole viral proteins is unable to elicit an efficient CTL response, interest has been directed towards designing vaccines based on defined epitope sequences. This is particularly the case with oncogenic viruses, since individual viral genes introduced in recombinant vectors have the potential to initiate tumorgenic processes. Two broad approaches are currently being considered to design an effective vaccine for controlling Epstein-Barr virus (EBV) associated diseases (for review see ref. (7)). These include directing immune responses to either EBV structural antigens or latent antigens.
In the last few years, most of the vaccine development efforts have concentrated on the use of a subunit preparation of gp350 (recombinant and affinity purified) and have been directed towards blocking virus attachment to the target cell in the oropharyrix (31). The general approach has been to immunize cotton-top marmosets with gp350 and determine their ability to restrict the outgrowth of EBV-positive lymphomas in these animals. Indeed, highly purified gp350, when administered subcutaneously in conjunction with adjuvants (muramyl dipeptide or ISCONIS). induced high levels of serum neutralizing antibodies and inhibited tumor formation in cotton-top tamarins (32). A number of recombinant vectors including, vaccinia-gp350 and adenovirus 5-gp350 have also been successfully used in these animals to block tumor outgrowth (33). The precise mechanism by which gp350 affords protection from lymphomas in cotton-top tamarins remains unclear. The fact that development of neutralizing antibody titres in vaccinated animals does not always correlate with protection indicates that gp350-specific T cell-mediated immune responses may also have an effector role (34). Furthermore, Yao and colleagues (35) showed that very low levels of neutralizing anti-gp350 antibodies are present in the saliva of healthy EBV-immune donors, which suggests that such antibodies are unlikely to be the basis of long-term immunity in healthy seropositive individuals. It has been postulated that gp350 specific T cell -mediated immune responses may have an effector role in protection. There has been no identification to date, however, of CTL epitopes within the EBV structural antigens.
Post-transplant lymphoproliferative disease (PTLD) that arises in organ transplant patients is an increasingly important clinical problem. Histological analysis of PTLD shows a quite complex clonal diversity ranging from polymorphic B lymphocyte hyperplasia to malignant monoclonal lymphoma. This range of pathology encompasses the collective term PTLD while the lymphomas are frequently referred to as immunoblastic lymphomas(IL). This condition is clearly associated with the proliferation of Epstein-Barr virus (EBV) infected B cells which are carried for life in all previously infected individuals (about 80% of adults and 20% of children 7 years) (45, 46, 47, 49). These EBV-infected B cells are normally restricted in their growth in vitro and in vivo by virus-specific cytotoxic T cells (CTLs) which recognise epitopes included within the EBV latent proteins (see below) (48). Immunosuppression inhibits these specific CTL and results in an expansion of the pool of EBV-infected B cells and the emergence of the clinical problems associated with PTLD. It is known that the individuals at greatest risk of PTLD are EBV seronegative recipients who receive a transplant from a seropositive donor (Crawford and Thomas, 1993). Immunisation of EBV seronegative graft recipients prior to engraftment will greatly reduce the risk of PTLD.
The role of the immune system in the rejection of virus-associated cancers has also been the subject of intense study recently. The hypothesis under investigation is that many neoplasms express viral antigens that should potentially enable them to be recognized and destroyed by the immune system, including both T helper cells and cytotoxic T lymphocytes (CTL). There is now compelling evidence that most of the Epstein-Barr virus (EBV)-associated malignancies escape this potent virus-specific CTL response by restricting viral gene expression (7.20,21). For malignancies such as nasopharyngeal carcinoma (NTC) and Hodgkin""s disease (HD). EBV nuclear antigen 1 (EBNA1) and latent membrane protein 1 (LMP1) are the only antigens consistently expressed and are therefore the potential target antigens for any future vaccine designed to control these tumors (3,28). Since it is well established that immunization with whole viral proteins does not elicit an efficient CTL response, interest has been directed towards developing peptide vaccines based on defined epitope sequences.
Results obtained by the present inventors indicate that CTL epitopes within EBV structural and latent proteins may be effective in providing antiviral immunity against EBV infection. In particular, the present inventors have analysed the latent antigen LMP1 sequence, using peptide stablization assays, and found that this antigen includes potential CTL epitopes. Following in vitro activation with these peptides, both polyclonal and clonal CTLs from HLA A2-positive donors showed strong reactivity against target cells expressing the LMP1 antigen. Moreover. lvmphoblastoid cell lines (LCL). expressing different HLA A2 supertypes were efficiently recognized by these CTLs, a result that has important implications for the design of an anti-viral vaccine aimed at protecting different ethnic populations.
The present inventors have also found that CTLs from acute infectious mononucleosis (IM) patients display strong reactivity against the EBV structural antigens gp85 and gp350. In addition, specific CTL epitopes within EBV structural antigens gp85 and gp350 have been identified for the first time. Importantly, prior immunisation of HLA A2/Kb transgenic mice with these gp350 and gp85 CTL epitopes induced a strong epitope-specific CTL response and afforded protection against gp85- or gp350-expressing vaccinia virus challenge. These results provide evidence, for the first time, of the existence of CTL epitopes in EBV structural proteins and show that they may be used for establishing strong anti-viral immunity against EBV infection.
Accordingly, in a first aspect the present invention provides a cytotoxic Epstein-Barr virus (EBV) T-cell epitope, the epitope being derived from an EBV structural antigen.
In a preferred embodiment of the first aspect of the present invention, the EBV structural antigen is gp85 or gp350.
In a second aspect the present invention provides a cytotoxic Epstein-Barr virus T-cell epitope, the epitope being selected from the group consisting of YLLEMLWRL (SEQ ID NO:1), YFLEILWGL (SEQ ID NO:32), YLLEILWRL (SEQ ID NO:33), YLQQNWWTL (SEQ ID NO:6), LLLALLFWL (SEQ ID NO:2), LLVDLLWLL (SEQ ID NO:3), LLLIALWNL (SEQ ID NO:4), WLLLFLAIL (SEQ ID NO:5), TLLVDLLWL (SEQ ID NO:7), LLWLLLFLA (SEQ ID NO:8), ILLIIALYL (SEQ ID NO:9), VLFIFGCLL (SEQ ID NO:10), RLGATIWQL (SEQ ID NO:11), ILYFIAFAL (SEQ ID NO:15), SLVIVTTFV (SEQ ID NO:17), LMIIPLINV (SEQ ID NO:20), TLFIGSHVV (SEQ ID NO:24), LIPETVPYI (SEQ ID NO:26), VLQWASLAV (SEQ ID NO:27) and QLTPHTKAV (SEQ ID NO:29).
In a third aspect the present invention provides a subunit vaccine including a cytotoxic Epstein-Barr virus (EBV) T-cell epitope according to the first aspect of the present invention.
In a preferred embodiment, the subunit vaccine includes at least one T-cell epitope selected from the group consisting of YLLEMILWRL (SEQ ID NO:1), YFLEILWGL (SEQ ID NO:32), YLLEILWRL (SEQ ID NO:33), YLQQNWWTL (SEQ ID NO:6). LLLALLFWL (SEQ ID NO:2). LLVDLLWLL (SEQ ID NO:3), LLLIALWNL (SEQ ID NO:4), WLLLFLAIL (SEQ ID NO:5), TLLVDLLWL (SEQ ID NO:7). LLWLLLFLA (SEQ ID NO:8), ILLIIALYL (SEQ ID NO:9), VLFIFGCLL (SEQ ID NO:10), RLGATIWQL (SEQ ID NO:11), ILYFIAFAL (SEQ ID NO:15). SLVIVTTFV (SEQ ID NO:17), LMIIPLINV (SEQ ID NO:20), TLFIGSHVV (SEQ ID NO:24). LIPETVPYI (SEQ ID NO:26). VLQWASLAV (SEQ ID NO:27) and QLTPHTKAV (SEQ ID NO:29).
In a preferred aspect of the present invention the epitope is selected from the group consisting of YLLEMLWRL (SEQ ID NO:1). YLQQNWWTL (SEQ ID NO:6). YFLEILWGL (SEQ ID NO:32), YLLEILWRL (SEQ ID NO:33). SLVIVTTFV (SEQ ID NO:17), LMIIPLINV (SEQ ID NO:20). TLFIGSHVV (SEQ ID NO:24), and VLQWASLAV (SEQ ID NO:27).
In a further preferred embodiment, the subunit vaccine includes one or more additional cytotoxic EBV T-cell epitopes. The additional cytotoxic EBV T-cell epitope(s) may be selected from those described in WO 97/45444, the entire contents of which are incorporated herein by reference.
In a further preferred form of the present invention the vaccine includes a water-in-oil formulation. It is further preferred that the vaccine includes at least one antigen to which the individual will mount an anamniestic response in addition to the at least one cytotoxic T-cell epitope.
The at least one antigen is preferably selected from the group consisting of tetanus toxoid, diphtheria toxoid, Bordetella pertussis antigens, poliovirus antigens, purified protein derivative (PPD), gp350 protein (Thorley-Lawson, D. A. and Poodry, C. A. (1982). Identification and isolation of the main component (gp35G-gp220) of Epstein-Barr virus responsible for generating neutralizing antibodies in vivo. J. Virol. 43, 730-736), helper epitopes and combinations thereof and is preferably tetanus toxoid.
It is preferred that the water-in-oil formulation is Montanide ISA 720. Additional information regarding this formulation can be found in WO 95/24926, the disclosure of which is incorporated herein by cross reference.
The subunit vaccine may also be formulated using ISCOMs. Further information regarding ISCOMs can be found in Australian Patent Nos. 558258, 590904, 632067, 589915, the disclosures of which are included herein by cross reference.
In a fourth aspect the present invention provides an isolated nucleic acid sequence encoding a cytotoxic Epstein-Barr virus (EBV) T-cell epitope according to the first aspect of the present invention.
In a preferred embodiment, the isolated nucleic acid sequence encodes at least one of the cytotoxic T-cell epitopes selected from the group consisting of YLLEMLWRL (SEQ ID NO:1), YFLEILWGL (SEQ ID NO:32), YLLEILWRL (SEQ ID NO:33). YLQQNWWTL (SEQ ID NO:6), LLLALLFWL (SEQ ID NO:2). LLVDLLWLL (SEQ ID NO:3), LLLIALWNL (SEQ ID. NO:4), WLLLFLAIL (SEQ ID NO:5), TLLVDLLWL (SEQ ID NO:7). LLWLLLFLA (SEQ ID NO:8), ILLIIALYL (SEQ ID NO:9), VLFIFGCLL (SEQ ID NO:10), RLGATIWQL (SEQ ID NO:11), ILYFIAFAL (SEQ ID NO:15), SLVIVTTFV (SEQ ID NO:17). LMIIPLINV (SEQ ID NO:20), TLFIGSHVV (SEQ ID NO:24), LIPETVPYI (SEQ ID NO:26), VLQWASLAV (SEQ ID NO:27) and QLTPHTKAV (SEQ ID NO:29).
As will be appreciated by those skilled in the field the nucleic acid sequence can be delivered as naked nucleic acid or using a suitable viral or bacterial vectors. Suitable bacterial vectors include the bacteria Salmonella spp. Suitable viral vectors include, for example, retroviral vectors, adenoviral vectors and vaccinia vectors. An example of a suitable vaccinia vector is a modified Vaccinia Ankara vector.
Vectors suitable for delivery of nucleic acid sequences have previously been described. For example, alphavirus vectors have become widely used in basic research to study thestructure and function of proteins and for protein production purposes. Development of a variety of vectors has made it possible to deliver foreignsequences as naked RNA or DNA, or as suicide virus particles produced using helper vector strategies. Preliminary reports also suggest that these vectors may be useful for in vivo applications where transient, high-level protein expression is desired, such as recombinant vaccines. The initial studies have already shown that alphavirus vaccines can induce strong humoral and cellular immune responses with good immunological memory and protective effects. See, for example, Tubulekas I., Berglund P., Fleeton M., and Liljestrom P. (1997) Alphavirus expression vectors and their use as recombinant vaccines:a minireview, Genie 190(1):191-195.
Recombinant pox viruses have been generated for vaccination against heterologous pathogens. Amongst these, the following are notable examples. (i) The engineering of the Copenhagen strain of vaccinia virus to express the rabies virus glycoprotein. When applied in baits, this recombinant has been shown to vaccinate the red fox in Europe and raccoons in the United States, stemming the spread of rabies virus infection in the wild. (ii) A fowlpox-based recombinant expressing the Newcastle disease virus fusion and hemagglutinin glycoproteins has been shown to protect commercial broiler chickens for their lifetime when the vaccine was administered at 1 day of age, even in the presence of maternal immunity against either the Newcastle disease virus or the pox vector. (iii) Recombinants of canarypox virus, which is restricted for replication to avian species, have provided protection against rabies virus challenge in cats and dogs against canine distemper virus, feline leukemia virus, and equine influenza virus disease. In humans, canarypox virus-based recombinants expressing antigens from rabies virus. Japanese encephalitis virus, and HV have been shown to be safe and immunogenic. (iv) A highly attenuated vaccinia derivative, NYVAC has been engineered to express antigens from both animal and human pathogens. Safety and immunogeniicity of NYVAC-based recombinants expressing the rabies virus glycoprotein, a polyprotein from Japanese encephalitis virus, and seven antigens from Plasmodium falciparum have been demonstrated to be safe and immunogenic in early human vaccine studies. See, for example, Paoletti E. (1996) Applications of pox virus vectors to vaccination:an update, Proc Natl Acad Sci U S A, 93(21):11349-11353.
Progress towards effective vaccines to control internal parasites, especially those affecting mucosal compartments, has been inhibited by the combined problems of the antigenic complexity of parasites and the lack of understanding of the host response. However, the accumulation of information regarding regulation of mucosal immunity has enabled a reappraisal of vaccination options to provide appropriate mucosal effector responses. The pivotal role of T cell influences, and in particular the contribution of cytokine signals, has been clearly established from in vitro studies, but data emerging from our laboratories provide evidence for these effects in vivo. We have demonstrated the role of T cells in determining the outcome of an intestinal response and propose a role for local Th2 cytokine production in this regard. To support this proposition, the distribution of cytokine mRNA has been determined by in situ hybridisation techniques in normal and parasitised animals. Further, we have shown that in the absence of Th2 cytokines (using gene knockout animals) mucosal responses are grossly deficient:we have also shown that this defect can be overcome by ,vector-directed gene therapy. These studies have indicated that new-mucosal immunisation opportunities exist by combining traditional immunisation approaches with strategies to upregulate local cytokine production. -However, the success of these new strategies will depend on selection of highly immunogenic subunit antigens, coupled with techniques for cytokine manipulation and delivery with appropriate adjuvant/vehicle formulations. This paper reviews delivery technologies available to chaperone labile antigenic and genetic material to appropriate sites for mucosal stimulation after systemic or oral administration. See, for example, Sutter G. et al (1994) A recombinant vector derived from the host range-restricted and highly attentuated MVA strain of vaccinia virus stimulates protective immunity in mice to influenza virus. Vaccine 12:1032-1040; and Husband A. J Bao S., McClure S. J., Emery D. L., Ramsay A. J. ( 1996) Antigen delivery strategies for mucosal vaccines. Int J Parasitol 8-9:825-834.
Attenuated Salmonella typhi vaccine strain CVD 908, which harbors deletion mutations in aroC and aroD, has been shown to be well-tolerated and highly immunogenic, eliciting impressive serum antibody, mucosal IgA and cell-mediated immune responses. A further derivative prepared by introducing a deletion in htrA (which encodes a heat-shock protein that also has activity as a serine protease in CVD 908 resulted in CVD 908-htrA. In phase 1 clinical trials, CVD 908-htrA appears very attractive as a live oral vaccine candidate. Both CVD 908 and CVD 908-htrA are useful as live vector vaccines to deliver foreign antigens to the immune system. Conditions that enhance the expression and immunogenicity of foreign antigens carried by CVD 908 and CVD 908-htrA are being investigated. For a review of Salmonella vectors, see Levine M. M., Galen J., Barry E., Noriega F., Chatfield S., Sztein M., Dougan G. And Tacket C (1996) Attenuated Salmonella as live oral vaccines against typhoid fever and as live vectors, J Biotechnol 44(1-3):193-196.
The isolated nucleic acid sequences may be in the form of nucleic acid vaccines. Further information regarding nucleic acid vaccines can be found in WO 96/03144 and in Suhrbier A (1997), Multi-epitope DNA vaccines, Immunol Cell Biol 75(4):402-408 the disclosures of which are incorporated herein by cross reference.
In a fifth aspect the present invention provides an isolated polypeptide, the polypeptide including at least one epitope according to the first or second aspects of the present invention.
The vaccines of the present invention may be used prophylactically or therapeutically.
The CTL epitopes of the present invention may be synthesised using techniques well known to those skilled in this field. For example, the CTL epitopes may be synthesised using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled xe2x80x9cPeptide Synthesisxe2x80x9d by Atherton and Sheppard which is included in a publication entitled xe2x80x9cSynthetic Vaccinesxe2x80x9d edited by Nicholson and published by Blackwell Scientific Publications. Preferably a solid phase support is utilised which may be polystyrene gel beads wherein the polystyrene may be cross-linked with a small proportion of divinylbenzene (e.g. 1%) which is further swollen by lipophilic solvents such as dichloromethane or more polar solvents such as dimethylformamide (DNIF). The polystyrene may be functionalised with chloromethyl or anionomethyl groups. Alternatively, cross-linked and functionalised polydiniethyl-acrylamide gel is used which may be highly solvated and swollen by DNIF and other dipolar aprolic solvents. Other supports can be utilised based on polyethylene glycol which is usually grafted or otherwise attached to the surface of inert polystyrene beads. In a preferred form, use may be made of commercial solid supports or resins which are selected from PAL-PEG, PAK-PEG, KA, KR or TGR.
In solid state synthesis, use is made of reversible blocking groups which have the dual function of masking unwanted reactivity in the a-amino, carboxy or side chain functional groups and of destroying the dipolar character of amino acids and peptides which render them inactive. Such functional groups can be selected from t-butyl esters of the structure RCOxe2x80x94OCMe3xe2x80x94COxe2x80x94NHR which are known as t-butoxy carbonyl or ROC derivatives. Use may also be made of the corresponding benzyl esters having the structure RCOxe2x80x94OCH2xe2x80x94C6Hs and urethanes having the structure C6H5CH2O COxe2x80x94NHR which are known as the benzyloxycarbonyl or Z-derivatives. Use may also be made of derivatives of fluorenyl methanol and especially the fluoreniyl-methoxy carbonyl or Fmoc group. Each of these types of protecting group is capable of independent cleavage in the presence of one other so that frequent use is made, for example, of BOC-benzyl and Fmoc-tertiary butyl protection strategies.
Reference also should be made to a condensing agent to link the amino and carboxy groups of protected amino acids or peptides. This may be done by activating the carboxy group so that it reacts spontaneously with a free primary or secondary amine. Activated esters such as those derived from p-nitrophenol and pentafluorophenyl may be used for this purpose. Their reactivity may be increased by addition of catalysts such as 1-hydroxybenzotriazole. Esters of triazine DHBT (as discussed on page 215-216 of the abovementioned Nicholson reference) also may be used. Other acylating species are formed in situ by treatment of the carboxylic acid (i.e. the Nxcex1-protected amino acid or peptide) with a condensing reagent and are reacted immediately with the amino component (the carboxy or C-protected amino acid or peptide). Dicyclohexylcarbodiimide, the BOP reagent (referred to on page 216 of the Nicholson reference), O""Benzotriazole-N, N, Nxe2x80x2Nxe2x80x2-tetra methyl-uronium hexaflurophosphate (HBTU) and its analogous tetrafluroborate are frequently used condensing agents.
The attachment of the first amino acid to the solid phase support may be carried out using BOC-anino acids in any suitable manner. In one method BOC amino acids are attached to chloromethyl resin by warming the triethyl ammonium salts with the resin. Fmoc-amino acids may be coupled to the p-alkoxybenzyl alcohol resin in similar manner. Alternatively, use may be made of various linkage agents or xe2x80x9chandlesxe2x80x9d to join the first amino acid to the resin. In this regard, p-hydroxymethyl phenylactic acid linked to aminomethyl polystyrene may be used for this purpose.
As will be readily appreciated by those skilled in the art the LMP1, gp85 and gp350 epitopes and vaccines of the present invention can be used to treat and to protect against EBV. Further, given the possible greater involvement of EBV infection in immnunocompromised individuals, the present invention may have particular application in the treatment and protection of individuals having decreased immune function, eg transplant patients. Importantly, the present inventors have found that EBV transformed lymphoblastoid cell lines expressing different HLA A2 supertypes are efficiently recognised by LMP1-specific CTL clones. This highlights the potential for the design of an antiviral vaccine aimed at treating and protecting different ethnic populations.
Accordingly, in a sixth aspect the present invention provides a method of preparing a composition for use in inducing CTLs in a subject, the method including admixing at least one epitope according to the first or second aspects of the present invention with at least one pharmaceutical acceptable carrier, diluent or excipient.
In a seventh aspect the present invention provides a method of reducing the risk of EBV infection in a subject which method includes administering to the subject an effective amount of:
(1) at least one CTL epitope according to the first or second aspects of the present invention;
(2) a subunit vaccine according to the third aspect of the present invention;
(3) a nucleic acid sequence according to the fourth aspect of the present invention:.
(4) a vector according to the fourth aspect of the present invention;
or
(5) a polypeptide according to the fifth aspect of the present invention.
In an eighth aspect the present invention provides a method of treating or preventing nasopharyngeal carcinoma or Hodgkin""s disease in a subject which method includes administering to the subject an effective amount of at least one CTL epitope derived from an EBV structural or latent antigen.
By xe2x80x9ceffective amountxe2x80x9d we mean a quantity of the epitope which is sufficient to induce or amplify a CTL response against an EBV antigen.
In a preferred embodiment of the eighth aspect of the present invention, the EBV structural antigen is gp83 or gp350 and the latent antigen is LMP1 or LMP2. In a further preferred embodiment the CTL epitope is an epitope as defined in the second aspect of the present invention.
The present inventors have also made the surprising finding that NPC cells which are recognised by CTL clones are subject to CTL lysis. This finding has important implications for the design of vaccines to control NPC tumours in vivo.
In an ninth aspect the present invention provides a method of treating or preventing growth of NPC or HD cells in a subject in need thereof which method includes administering to the subject at least one CTL epitope derived from an EBV structural or latent antigen.
In a preferred embodiment of the ninth aspect of the present invention, the EBV CTL epitopes are derived from the gp85 or gp350 -antigens. In a further preferred embodiment, the EBV CTL epitopes are derived from the LMP1 or LMP2 antigens. Preferably, the CTL epitopes are derived from the LMP1 antigen.
In a tenth aspect the present invention provides a method of treating or preventing the growth of NPC or HD) cells in a first subject which method includes transferring to the subject EBV-specific CTLs which recognise NPC or HD cells.
In a preferred embodiment the EBV-specific CTLs are obtained from NPC patients by in vitro stimulation of CTLs by exposure to EBV CTL epitopes. Alternatively, the EBV-specific CTLs may be obtained from a second subject, wherein the second subject is infected with EBV but does not have NPC.
In a further preferred embodiment of the tenth aspect of the present invention, the EBV-specific CTLs are LMP1 and/or LMP2-specific CTLs.
In an eleventh aspect the present invention provides a method of reducing the risk of infectious mononucleosis or post transplantation lymphoproliferative disease in a subject which method includes administering to the subject an effective amount of:
(1) at least one CTL epitope according to the first or second aspects of the present invention;
(2) a subunit vaccine according to the third aspect of the present invention;
(3) a nucleic acid sequence according to the fourth aspect of the present invention;
(4) a vector according to the fourth aspect of the present invention;
or
(5) a polypeptide according to the fifth aspect of the present invention.
The terms xe2x80x9ccomprisexe2x80x9d, xe2x80x9ccomprisesxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d as used throughout the specification are intended to refer to the inclusion of a stated component or feature or group of components or features with or without the inclusion of a further component or feature or group of components or features.
In order that the nature of the present invention may be more clearly understood forms thereof will now be described with reference to the following examples and figures.